The present invention is directed to the production of new derivatives of streptomycin by a technique called "mutational biosynthesis". This technique is disclosed in U.S. patent application Ser. No. 3,669,838 entitled Method of Making Antibiotics Containing the Aminocyclitol Subunit by Shier et al. A brief discussion of mutational biosnthesis appears below.
A culture ordinarily makes an antibiotic (ABC) composed of three moieties (A,B,C) from simple carbon and nitrogen sources: ##STR1##
A mutant is obtained that cannot make A; thus, it cannot make the antibiotic ABC. If, however, A is fed in the medium, it can make the antibiotic. ##STR2##
If instead of A, an analogue (A') is fed into the medium, the mutant can produce a new antibiotic (A'BC). Of course, the foregoing depends on the assumptions that:
A' will go into the cell; the incorporating enzyme is not too specific; and, A'BC has antibiotic activity.
At this point, it should be noted that the antibiotics disclosed in the Shier et al. patent referred to above, contain 2-deoxystreptamine as its aminocyclitol.
Streptomycin is a different antibiotic in that it contains streptidine. It should also be noted that streptidine is most accurately characterized as a guanidinocyclitol rather than an aminocyclitol. The structural formulas for 2-deoxystreptamine and streptidine appear below. ##SPC1##
The significance of the foregoing is that prior to the present invention, mutational biosynthesis had not been successful with antibiotics containing streptidine or analogues of streptidine. Indeed, in a doctorial thesis which can be obtained from the University of Illinois, Urbana, Ill., entitled The Hybrimycins by W. T. Shier, the failure to produce the mutant required for streptomycin production by mutational biosynthesis is reported.
Mutational biosynthesis has been tried with Bacillus circulans, the producer of butirosins. This is another group of two antibiotics containing deoxystreptamine. Workers have also been successful in obtaining a deoxystreptamine mutant which could use streptamine and streptidine to form new antibiotics (DeFuria and Claridge, Abstr. Ann. Mtg. Amer. Soc. Microbiol., Miami Beach p. 13, 1973; Claridge et al., Devel. Industr. Microbiol. 15, 101,1974)
There are similar studies on Streptomyces kanamyceticus, the producer of kanamycin, and on Streptomyces ribosididicus, the producer of ribostamycin. There are also deoxystreptamine-containing antibiotics. The deoxystreptamine mutant of S. ribosidificus could use 1-N-methyl-deoxystreptamine, streptamine, 2-epi-streptamine, and 3',4'-dideoxyneamine to make new active ribostamycin analogues containing these modified aminocyclitols. The S. kanamyceticus mutant could use 1-N-methyl-deoxystreptamine and epi-streptamine. These studies are described in Kojima and Satoh, J. Antibiotics 26, 784 (1973).
In Biochem. Biophys. Acta 148, 335 (1967) a study of the biosynthesis of streptomycin is disclosed and a cell-free enzymatic activity from S. griseus which phosphorylated streptidine in the presence of ATP is reported. The enzyme is also reported to be active on 2-deoxystreptidine, but, inactive on N-amidinostreptamine, N'-amidinostreptamine, N-amidinoinosamine and myoinositol. It was not known whether the enzyme had any role in streptomycin formation. It is also reported (Walker, Devl. Industr. Microbiol. 8, 109, 1967) that intact mycelia of all species of Streptomyces tested (except one) could phosphorylate streptidine even though many were not streptomycin producers. It has been suggested that such streptomycin non-producing species might possibly make streptomycin if fed streptidine or streptomycin analogues. It should be noted that the use of mutants of S. griseus in a mutational biosynthesis process has never been suggested. In J. Bacteriol. 99, 401 (1969) it was noted that the enzyme from S. griseus which phosphorylated streptidine and 2-deoxystreptidine also phosphorylated streptomycin at the same 6 position of streptidine and that the real function of the enzyme might be to inactivate streptomycin or to maintain it in an inactive form in the mycelium. The significance of the foregoing is that it has been known that deoxystreptidine acts as a substrate for an enzyme acting on streptidine.